This invention relates to methods for producing plants that genetically clone themselves through their own seed (gametophytic apomicts) from plants that normally reproduce sexually. More particularly, the invention relates to processes that include (a) selection of two or more sexual lines that express reproductive phenotypes divergent from each other, which may in some cases require plant breeding and selection to obtain sufficient degrees of divergence, (b) hybridization among plants divergent in reproductive phenotype, and (c) amphiploidization (doubling of chromosomes) either before or after hybridization.
It is likely that apomixis has a greater potential for increasing yields of food, feed, and fiber than any other plant mechanism. Apomixis occurs in about 0.3% of flowering plant species. The present patent application describes methods for making sexual plants apomictic without crossing them to wild apomictic relatives or using mutagenic procedures, both of which have been attempted but with disappointing results. The procedures described herein mimic how apomixis evolved in nature (J. G. Carman, Asynchronous Expression of Duplicate Genes in Angiosperms May Cause Apomixis, Bispory, Tetraspory, and Polyembryony, 61 Biol. J. Linnean Soc. 51-94 (1997) (incorporated herein by reference; hereinafter, “Linnean”), and enable persons skilled in the art of plant breeding and genetics to convert inbred crops, including wheat, barley, and rice, into apomictic hybrid crops with potential yield increases of 10 to 30% over currently used inbred varieties. Crops currently used as hybrids, such as maize, may also be made apomictic. Apomictic hybrids of either inbred or typically hybrid crops will behave as hybrids only in terms of their superior yields. The seed of apomictic hybrids are genetic clones of the mother hybrid, i.e., genetic segregation does not occur. Thus, farmers could use a small fraction of their harvest for seed and expect high yields and uniformity year after year. This would allow hybrids, the seed of which is typically very expensive, to be used in impoverished nations for the first time, which could contribute substantially to another “green revolution.”
Gregor Mendel conducted, unknowingly, the first genetic experiments with gametophytic apomicts (plants that produce seed asexually). He reported the successful crossing of different Hieracium lines but commented on the extreme difficulty of preventing self fertilization. What he thought was high frequency accidental selfing (in his facultatively-apomictic Hieracium lines) was actually high frequency apomictic seed formation. To add to his frustration, Mendel failed to observe segregation among the F2s of the few F1s he managed to produce. His F2s were actually apomictic clones of his F1s, and they invariably expressed their respective F1 phenotypes (S. E. Asker & L. Jerling, Apomixis in Plants (CRC Press, 1992) (hereby incorporated by reference; hereinafter, “Asker & Jerling”).
Several thousand species of Hieracium had been described by the time Mendel hybridized members of this agamic complex. This pronounced polymorphy, and that observed in other agamic complexes (Antennaria, Erigeron, Taraxacum, Potentilla), coupled with Mendel's results in producing new polymorphs by crossing facultative apomicts, led early geneticists to conclude that hybridization in agamic complexes is a major mechanism of speciation. Evidence for this conclusion is replete, e.g., in Amelanchier and Crataegus (C. S. Campbell & T. A. Dickinson, Apomixis, Patterns of Morphological Variation, and Species Concepts in Subfam. Maloideae (Rosaceae), 15 Systematic Bot. 124-25 (1990) (incorporated herein by reference), in Antennaria (Bayer et al., Phylogenetic Inferences in Antennaria (Asteraceae: Gnaphalieae) Based on Sequences from the Nuclear Ribosomal DNA Internal Transcribed Spacers (ITS), 83 Amer. J. Bot. 516-527 (1996) (incorporated herein by reference), in numerous agamic grass complexes (E. A. Kellogg, Variation and Species Limits in Agamospermous Grasses, 15 Systematic Bot. 112-23 (1990) (incorporated herein by reference), in Rubus (Nybom, Evaluation of Interspecific Crossing Experiments in Facultatively Apomictic Blackberries (Rubus Subgen. Rubus) Using DNA Fingerprinting, 122 Hereditas 57-65 (1995) (incorporated herein by reference), in Taraxacum (Richards, The origin of Taraxacum agamospecies, 66 Biol. J. Linnean Soc. 189-211 (1973) (incorporated herein by reference), and others. In contrast, two conflicting opinions soon developed among early geneticists regarding the role of hybridization in the origins of apomixis. Strausburger, Zeitpunkt der Bestimmung des Geschlechtes, Apogamie, Parthenogenesis und Reduktionsteilung, 7 Hist. Beitr. 1-124 (1909) (incorporated herein by reference), Ostenfeld, Experiments on the Origin of Species in the Genus Hieracium (Apogamy and Hybridism), 11 New Phytol. 347-54 (1912) (incorporated herein by reference), and Holmgren, Zytologische Studien über die Fortpflanzung bei den Gattungen Erigeron und Eupatorium, 59 Kgl. Sven Vetenskapsakad. Ak. Handl. No. 7, 1-118 (1919) (incorporated herein by reference) believed apomixis is controlled by genetic factors (genes) specific to apomixis and is not a consequence of hybridization. In contrast, A. Ernst, Bastardierung als Ursache der Apogamie im Pflanzenreich, Fischer, Jena (1918) (incorporated herein by reference), believed that the cytological anomalies of reproduction responsible for apomixis are extensions of the genomic disturbances observed in wide hybrids.
Ernst amassed much evidence to support his hybridization hypothesis, which included the facts that apomicts have high chromosome numbers (they are generally polyploid), that agamic complexes are highly polymorphic, and that the sex cells of apomicts often degenerate in a manner observed in interspecific hybrids. A major tenet of Ernst's hypothesis, and the one which soon caused its widespread dismissal (and continues to cause its legitimate dismissal today), was that hybrids form a continuum from fully functional sexual reproduction, to apomixis, and finally to vegetative reproduction. Where a hybrid fit on the continuum depended on how closely related the parent species are, e.g., if the parents are closely related, the hybrid will reproduce sexually, if the parents are moderately related, the hybrids may tend to be apomictic, if the parents are distantly related, the hybrids may tend to reproduce by vegetative propagation. Thus, according to Ernst, apomixis arises only in wide hybrids. Ernst did not identify mechanisms to support a hybrid origin for apomixis other than the wideness of the cross.
Ernst's hypothesis received support in the 1920s and 1930s (Harrison, The Inheritance of Melanism in Hybrids Between Continental Tephrosia crepuscularia and Britisht bistortata, with Some Remarks on the Origin of Parthenogenesis in Interspecific Crosses, 9 Genetika 4467 (1927) (incorporated herein by reference); G. L. Stebbins, Cytology of Antennaria. II. Parthenogenetic Species, 94 Bot. Gaz. 322-45 (1932) (incorporated herein by reference)), but most geneticists had rejected it by the time Åke Gustafsson published his comprehensive treatise, Å Gustafsson, Apomixis in Higher Plants, I. The Mechanism of Apomixis, 42 Lunds Universitets Årsskrift 1-67 (1946); Å Gustafsson, Apomixis in Higher Plants, II. The Causal Aspect of Apomixis, 43 Lunds Universitets Årsskrift 69-179, (1947); Å Gustafsson, Apomixis in Higher Plants, III. Biotype and Species Formation, 43 Lunds Universitets Årsskrift 181-370 (1947) (incorporated herein by reference). In this treatise, Gustafsson concluded: “In no case is it proved that hybridization itself has been able to produce apomixis. On the contrary, it is certain that the apomictic method of reproduction has in many cases arisen within a species population.” The fact that some apomicts are autopolyploid, which was well documented by 1946, legitimately squelched any perceived requirement for wide hybridization. Hence, Ernst's hypothesis collapsed because it claimed that the cytological mechanisms of apomixis are extensions of the cytological abnormalities observed during gamete formation in wide hybrids, which, by definition, contain grossly divergent genomes that prevent normal chromosome pairing during meiosis. We know today that this is not the case, i.e., chromosome pairing in many apomicts is normal. Since Gustafsson's treatise, few geneticists have suggested that the role of hybridization in agamic complexes exceeds that of speciation among taxa already containing a genetically-determined predisposition for apomixis. Dissecting this genetic predisposition is being attempted but is proving to be a formidable task.
Few genetic analyses of apomixis were conducted prior to Gustafsson's treatise, and these lacked the numbers of progeny required to draw specific conclusions (Asker & Jerling). Nevertheless, they suggested to Gustafsson that apomixis is caused by interbalanced systems of recessive genes. Gustafsson defended this view by citing examples in (a) Parthenium, Poa, and Potentilla, where embryo sac formation and parthenogenesis are under independent genetic control, and (b) Poa, Potentilla, and Rubus, where hybrids between two apomicts or between an apomict and a sexual parent are either sexual or apomictic with no clear pattern as to the outcome (suggestive of many recessive genes). In Rubus, sexual F1s had been produced from apomictic parents, and these F1s produced a low percentage of apomictic F2s, which again suggested segregation for multiple recessive factors.
Another realm of apomixis research that has produced ambiguities involves the effects of artificially changing the ploidy of apomicts. The general trend is for apomixis to intensify when the ploidy of an apomict is artificially increased. However, exceptions in Potentilla, Taraxacum, Paspalum, and Poa have been found in which artificially-induced increases in ploidy cause (a) haploparthenogenesis, in which reduced eggs form and develop without fertilization, (b) BIII hybridization, in which unreduced eggs are fertilized, and (c) complete restoration of sexuality. Sexuality has also been restored in apomictic Poa by haploidization. Concerning such ambiguities, Asker and Jerling concluded: “Our difficulties in explaining the ‘breakdown of apomixis’ remain connected with our ignorance concerning [its] regulation . . . ” Such ambiguities persuaded Gustaffson to reject simple dominance models for the inheritance of apomixis. He saw little evidence for them and was unconvinced by such claims in Dryopteris, Hieracium, Hypericum, Potentilla, and Sorbus. In each case, Gustaffson provided reasonable alternatives for the published claims.
Distorted segregation ratios can also hinder genetic analyses of apomixis. Certain apomicts in Dicanthium and Themeda tend to be sexual when grown in long days and apomictic when grown in short days (Asker & Jerling). Nevertheless, replicated studies with consistent segregation ratios have now been conducted in Panicum (Asker & Jerling), Tripsacum (O. Leblanc et al., Detection of the Apomictic Mode of Reproduction in Maize-Tripsacum Hybrids Using Maize RFLP Markers, 90 Theor. Appl. Genet. 1198-1203 (1995) (incorporated herein by reference), and Brachiaria (Valle & Miles, Breeding of Apomictic Species, in Y. Savidan & J. G. Carman, Advances in Apomixis Research (FAO, Rome, in press) (incorporated herein by reference), and each study suggested that apomeiosis (detected cytologically) is controlled by a single dominant allele. However, other recent studies challenge this conclusion, e.g., the apomeiosis “allele” in the Tripsacum accession studied by O. Leblanc et al., 90 Theor. Appl. Genet. 1198-1203 (1995), is part of a large linkage group in which recombination is suppressed, and a similar linkage group appears to exist in apomictic Pennisetum (Grimanelli et al., Molecular Genetics in Apomixis Research, in Y. Savidan, J. G. Carman, Advances in Apomixis Research (FAO, Rome, 1998) (in press, incorporated herein by reference)). These linkage groups may contain multiple genes required for apomeiosis (Grimanelli et al., Mapping Diplosporous Apomixis in Tetraploid Tripsacum: One Gene or Several Genes?, Heredity (1998) (in press, incorporated herein by reference)).
Two research groups are presently attempting to introgress apomixis into maize from Tripsacum, and neither has reported its expression in addition lines with less than nine Tripsacum chromosomes. In one group, two apomictic maize triploids containing nine Tripsacum chromosomes (3x+9) were produced. Cytogenetic and molecular studies indicated that the nine Tripsacum chromosomes in each line were probably the same (B. Kindiger et al., Evaluation of Apomictic Reproduction in a Set of 39 Chromosome Maize-Tripsacum Backcross Hybrids, 36 Crop Sci. 1108-13 (1996) (incorporated herein by reference)). A third triploid addition line, again with nine Tripsacum chromosomes (3x+9), was produced by the same group. However, many of the nine chromosomes in this line differed from the nine chromosomes of the two former lines. The maize chromosomes were the same for all three lines. The latter 3x +9 plant was also apomictic, but the frequency of apomixis was only 10 to 15%, compared with 95 to 100% for the two former lines (Sokolov et al, Perspectives of Developing Apomixis in Maize, Priority Directions of Genetics, Russia (1997) (progress report; incorporated herein by reference)). These data, and unpublished findings from the other group attempting to transfer apomixis to maize (Grimanelli et al., Molecular Genetics in Apomixis Research, in Y. Savidan, J. G. Carman, Advances in Apomixis Research (FAO, Rome, 1998) (in press)), suggest a complex mode of inheritance for apomixis. In another study, sexual T. dactyloides diploids were crossed with highly apomictic T. dactyloides triploids to produce aneuploids. All but three of 46 F1s showed tendencies for apomeiosis (determined cytologically). However, the highly apomeiotic F1s contained seven or more additional chromosomes (above the diploid level), and all F1s with chromosome numbers near the diploid level were sexual (Sherman et al., Apomixis in Diploid X Triploid Tripsacum dactyloides Hybrids, 34 Genome 528-32 (1991) (incorporated herein by reference)), which suggests complex inheritance. Finally, apomixis in artificially produced Tripsacum triploids cosegregated with five Tripsacum linkage groups syntenic with regions from three maize chromosomes (Blakey et al, Co-segregation of DNA Markers with Tripsacum Fertility, 42 Maydica 363-69 (1997) (incorporated herein by reference)), which further discredits a simple inheritance mechanism for the control of apomixis (at least when attempting to transfer the apomixis mechanism to other species or other lines within a species). It is reasonable to assume that a major gene (a regulatory or controlling gene) could prevent apomixis from occurring when in the recessive condition, thus making apomixis appear to be under simple genetic control. However, such gene(s) belong to many genes required for the expression of apomixis and will not confer apomixis to other species by themselves (Linnean). The studies just reviewed infer: (a) multiple genes are required for apomixis, (b) genes affecting facultativeness may behave additively, (c) some Tripsacum chromosomes affect facultativeness more than others, and (d) alleles from at least three maize chromosomes fail to substitute for their homeologous (syntenic) counterparts from Tripsacum in conferring apomixis.
Meiotic mutants are central to the simple inheritance hypotheses (Linnean). Recent mutation hypotheses suggest apomixis is not expressed unless appropriate meiotic mutations are combined with an appropriate polygenic predisposition (Mogie, The Evolution of Asexual Reproduction in Plants (1992) (incorporated herein by reference); Grimanelli et al, Molecular Genetics in Apomixis Research, in Y. Savidan, J. G. Carman, Advances in Apomixis Research (FAO, Rome, 1998) (in press)). However, recently obtained evidence indicates that the alleles thought to be mutations are actually part of the polygenic predisposition and are required for sexual reproduction in marginal habitats. What has not been previously appreciated, but which is shown herein, is that it is the union of divergent ecotypes (interracial or interspecific) through secondary contacts that permits apomixis to arise (see also Linnean).
In view of the foregoing, it will be appreciated that providing methods for producing apomictic plants would be a significant advancement in the art.